Pt-CeO2 coating of carbon nanotubes grown on anode gas diffusion layer of the polymer electrolyte membrane fuel cell.

The growing of carbon nanotubes on a gas diffusion layer (GDL) was investigated using electron microscopy and photoelectron spectroscopy. The 30 nm thick Pt doped CeO2 layers were deposited by (rf) magnetron sputtering using a CeO2-Pt target on a carbon diffusion layer overgrown by carbon nanotubes. The anode prepared in such a way was tested in the proton exchange membrane fuel cell. Hydrogen/air fuel cell activity measurements normalized to the amount of used Pt revealed high specific power (W mg(-1) Pt). The high activity of this anode with CNT-grown is explained by high specific area of the catalyst, high conductivity of CNT-GDL junction and high activity of platinum present in cationic state Pt2,4+. Very high specific power and low cost together with physical vapor deposition of catalyst makes this anode preparation promising for micro fabrication of fuel cells to power mobile systems.

Platinum-doped CeO2 thin film catalysts prepared by magnetron sputtering.

The interaction of Pt with CeO(2) layers was investigated by using photoelectron spectroscopy. The 30 nm thick Pt doped CeO(2) layers were deposited simultaneously by rf-magnetron sputtering on a Si(001) substrate, multiwall carbon nanotubes (CNTs) supported by a carbon diffusion layer of a polymer membrane fuel cell and on CNTs grown on the silicon wafer by the CVD technique. The synchrotron radiation X-ray photoelectron spectra showed the formation of cerium oxide with completely ionized Pt(2+,4+) species, and with the Pt(2+)/Pt(4+) ratio strongly dependent on the substrate. The TEM and XRD study showed the Pt(2+)/Pt(4+) ratio is dependent on the film structure.

Rats with monosodium glutamate-induced obesity and insulin resistance exhibit low expression of Galpha(i2) G-protein.

In order to test the potential role of inhibitory G-proteins in mechanisms of insulin resistance in adipose tissue of obese animals we determined the content of Galpha(i1) and Galpha(i2) proteins and an extent of protein tyrosine phosphorylation in epididymal fat tissue cell membranes using immunoblot. Monosodium glutamate-induced obese rats displayed adipose tissue hypertrophy, elevated levels of insulin, leptin and slightly elevated serum glucose. We found significantly decreased protein content of Galpha(i2) in adipose tissue plasma membranes of obese rats. This was in accordance with lower protein tyrosine phosphorylation noticed in adipose tissue cell homogenate of glutamate-treated animals. Our results confirm the role of Galpha(i2) in development of insulin resistance by crosstalk between the reduced level of inhibitory G-protein and insulin receptor mediated most likely by activation of phosphotyrosine protein dephosphorylation.

Evaluation of serum and urine clusterin as a potential tumor marker for urinary bladder cancer.

Clusterin is a stress-associated cytoprotective chaperone up-regulated by various apoptotic triggers in many cancers and neurodegenerative diseases. No valid information about serum or urine clusterin concentration in patiens with bladder cancer exists. Aim of our paper was evaluation of the urine and serum clusterin concentrations in individuals with bladder cancer. Blood and urine samples were used from 43 patients with urothelial tumors of the urinary bladder and from 50 patients with benign urological diseases. Blood and urine were collected before cystoscopy. Enzyme-linked immunosorbent assays (ELISA) were performed for clusterin from serum and urine. Serum clusterin was higher in individuals with bladder cancer (means 185,812.5 vs 171,946.5 kU/l, p=0.04). Sensitivity for bladder cancer detection was 73% and specificity 55% (AUC 0.63); efficacy was not sufficient. Urine values of clusterin were higher in individuals with bladder cancer (197.2 vs 67.7, p=0.0007). Sensitivity for bladder cancer detection was 49% and specificity 92% (AUC 0.75, LR+ 6.1, PPV+ 84%); diagnostic efficacy was sufficient. In conclusion, serum and urine clusterin can differ between bladder cancer patients and the control group. Urine clusterin could be the possible laboratory marker of bladder cancer. Further research is warranted to confirm findings in larger studies of various clinical status.

P-glycoprotein-mediated multidrug resistance phenotype of L1210/VCR cells is associated with decreases of oligo- and/or polysaccharide contents.

Multidrug resistance of murine leukaemic cell line L1210/VCR (obtained by adaptation of parental drug-sensitive L1210 cells to vincristine) is associated with overexpression of mdr1 gene product P-glycoprotein (Pgp)-the ATP-dependent drug efflux pump. 31P-NMR spectra of L1210 and L1210/VCR cells (the latter in the presence of vincristine) revealed, besides the decrease of ATP level, a considerable lower level of UDP-saccharides in L1210/VCR cells. Histochemical staining of negatively charged cell surface binding sites (mostly sialic acid) by ruthenium red (RR) revealed a compact layer of RR bound to the external coat of sensitive cells. In resistant cells cultivated in the absence or presence of vincristine, the RR layer is either reduced or absent. Consistently, resistant cells were found to be less sensitive to Concanavalin A (ConA). Moreover, differences in the amount and spectrum of glycoproteins interacting with ConA-Sepharose were demonstrated between sensitive and resistant cells. Finally, the content of glycogen in resistant cells is lower than in sensitive cells. All the above facts indicate that multidrug resistance of L1210/VCR cells mediated predominantly by drug efflux activity of Pgp is accompanied by a considerable depression of oligo- and/or polysaccharides biosynthesis.

Solution structure of mithramycin dimers bound to partially overlapping sites on DNA.

Mithramycin (MTH) is a DNA-binding antitumor agent containing A-B disaccharide and C-D-E trisaccharide segments projecting from opposite ends of an aglycone chromophore. We have previously reported on the solution structure of the MTH-DNA 6-mer complex based on a combined NMR and molecular dynamics study. This study established that the Mg(2+)-coordinated mithramycin dimer bound to a widened minor groove centered about the sequence-specific (G-C).(G-C) site and that the C-D-E trisaccharide segments from individual monomers were directed towards opposite ends of the helix spanning a six base-pair segment. This research is now extended to the binding of mithramycin dimers to partially overlapping sites on the self-complementary d(T-A-G-C-T-A-G-C-T-A) 10-mer duplex. The six base-pair mithramycin dimer footprint centered about (G-C).(G-C) steps should result in a potential steric clash in the center of the helix involving the inwardly pointing E-sugars of the pair of mithramycin dimers bound to the DNA 10-mer duplex. The MTH-d(T-A-G-C-T-A-G-C-T-A) complex (two MTH dimers per duplex) yields narrow and well-resolved NMR spectra, which have been assigned to identify intramolecular and intermolecular nuclear Overhauser enhancement (NOE) connectivities in the complex. The solution structure of the MTH-DNA 10-mer complex based on distance-restrained molecular dynamics calculations has defined the conformation of the drug and the DNA necessary for accommodation of the pair of mithramycin dimers on the DNA 10-mer helix. Specifically, the inwardly pointing E-sugars retain their face-down alignment towards the floor of the minor groove and occupy adjacent binding sites in the center of the duplex. This is achieved, in part, through torsion angle differences in the glycosidic linkage bonds along the length of the inwardly pointing aglycone-C-D-E trisaccharide segment relative to its outwardly pointing aglycone-C-D-E trisaccharide counterpart in the complex. In addition, a pronounced kink at the central (T-A).(T-A) step opens the minor groove and generates additional space to accommodate the inwardly pointing E-sugars at adjacent sites in the MTH-DNA 10-mer complex. These studies establish conformational plasticity in the C-D-E trisaccharide segment of the mithramycin dimer and deformability of the DNA helix allowing mithramycin dimers to bind to partially overlapping minor groove sites on the DNA helix.

Solution structure of the monoalkylated mitomycin C-DNA complex.

Mitomycin C (MC) is a potent antitumor antibiotic which alkylates DNA through covalent linkage of its C-1" position with the exocyclic N2 amino group of guanine to yield the [MC]dG adduct at the duplex level. We report on the solution structure of the monoalkylated MC-DNA 9-mer complex where the [MC]dG5 adduct is positioned opposite dC14 in the d(A3-C4-[MC]G5-T6).d(A13-C14-G15-T16) sequence context. The solution structure was solved based on a combined NMR-molecular dynamics study including NOE intensity based refinement. The formation of the [MC]dG adduct occurs with retention of the Watson-Crick alignment at the [MC]dG5.dC14 base-pair and flanking pairs in the complex. The MC ring is positioned in the minor groove with its indoloquinone aromatic ring system at a approximately 45 degrees angle relative to the helix axis and directed towards the 3'-direction on the unmodified strand. The MC indoloquinone chromophore is asymmetrically positioned in a slightly widened minor groove so that its plane is parallel to and stacked over the d(C14-G15-T16) segment on the unmodified strand with its other face exposed to solvent. The MC five-membered ring adopts an envelope pucker with its C-2" atom displaced from the mean plane and directed away from the unmodified strand. We observe conformational perturbations in the DNA 9-mer duplex on formation of the monoalkylated MC complex. Specifically, the base-pairs are displaced by approximately -3.0 A towards the major groove on positioning the MC in the minor groove. This perturbation is accompanied by base stacking patterns similar to those observed in A-DNA while the majority of the sugars adopt puckers characteristic of B-DNA. Conformational perturbations as monitored by helix twist, sugar pucker pseudorotation and glycosidic torsion angles are also observed for the d(T6-C7-I8).d(C11-G12-A13) segment that is adjacent to but does not overlap the MC binding on the 9-mer duplex. We note that the O-10" atom on the carbamate side-chain of MC forms an intermolecular hydrogen bond with the exocyclic amino group of dG15 in two of the three refined structures of the complex. The solution structure of the complex containing this intramolecular hydrogen bond readily explains both the previously observed d(C-G).d(C-G) sequence requirement for cross-linking and the observed, somewhat less stringent, requirement of the same sequence for the initial monoalkylation step.(ABSTRACT TRUNCATED AT 400 WORDS)

Molecular recognition in the FMN-RNA aptamer complex.

We report on a combined NMR-molecular dynamics calculation approach that has solved the solution structure of the complex of flavin mononucleotide (FMN) bound to the conserved internal loop segment of a 35 nucleotide RNA aptamer identified through in vitro selection. The FMN-RNA aptamer complex exhibits exceptionally well-resolved NMR spectra that have been assigned following application of two, three and four-dimensional heteronuclear NMR techniques on samples containing uniformly 13C, 15N-labeled RNA aptamer in the complex. The assignments were aided by a new through-bond NMR technique for assignment of guanine imino and adenine amino protons in RNA loop segments. The conserved internal loop zippers up through the formation of base-pair mismatches and a base-triple on complex formation with the isoalloxazine ring of FMN intercalating into the helix between a G.G mismatch and a G.U.A base-triple. The recognition specificity is associated with hydrogen bonding of the uracil like edge of the isoalloxazine ring of FMN to the Hoogsteen edge of an adenine at the intercalation site. There is significant overlap between the intercalated isoalloxazine ring and its adjacent base-triple platform in the complex. The remaining conserved residues in the internal loop participate in two G.A mismatches in the complex. The zippered-up internal loop and flanking stem regions form a continuous helix with a regular sugar-phosphate backbone except at a non-conserved adenine, which loops out of the helix to facilitate base-triple formation. Our solution structure of the FMN-RNA aptamer complex is to our knowledge the first structure of an RNA aptamer complex and outlines folding principles that are common to other RNA internal and hairpin loops, and molecular recognition principles common to model self-replication systems in chemical biology.

Saccharide-RNA recognition in an aminoglycoside antibiotic-RNA aptamer complex.

BACKGROUND: Aminoglycoside antibiotics are known to target ribosomal, retroviral and catalytic RNAs with high affinity and specificity. Recently, in vitro selection experiments have identified RNA aptamers that bind to aminoglycoside antibiotics with nanomolar affinity and stringent specificity, allowing discrimination between closely related family members. There has, to date, been limited structural information on the molecular basis of such saccharide-RNA recognition. RESULTS: We describe a solution-structure determination of the tobramycin-RNA aptamer complex, obtained using NMR and molecular dynamics. The structure gives insight into the molecular features associated with saccharide-RNA recognition. Tobramycin adopts a defined alignment and binds to the RNA major groove centered about a stem-loop junction site. A portion of the bound tobramycin is encapsulated between the floor of the major groove and a looped-out cytosine residue that forms a flap over the binding site in the complex. CONCLUSIONS: The emergence of antibiotic-resistant pathogens and their impact on human health continues to be a major concern in the medical community. Rational modification of existing antibiotics aimed at improving their efficacy requires a molecular view of their receptor-binding sites. We have provided such a molecular view for a member of the aminoglycoside antibiotic family that targets RNA.

One-year results from cryopreserved mitral allograft transplantation into the tricuspid position in a sheep experimental model.

Mitral allografts are still used only exceptionally in the mitral or tricuspid position. The main indication remains infectious endocarditis of atrioventricular valves for its flexibility and low risk of infection. The aim of our study was to evaluate 1-year results of mitral allografts transplantation into the tricuspid position in a sheep model. Mitral allografts were processed, cryopreserved, and transplanted into the tricuspid position anatomically (Group I - 11 animals) or antianatomically (Group II - 8 animals). All survivors (4 from Group I, and 3 from Group II) were checked at 3, 6, and 12 months by echocardiography with the exception of one survivor from Group II (which was examinated only visually). Examination throughout follow-up included for mitral allograft regurgitation and annuli dilatation. At postmortem, the papillary muscles were healed and firmly anchored to the right ventricular wall in all subjects. Transventricular fixation of the papillary muscles with buttressed sutures was proven to be a stable, reproducible, and safe method for anchoring mitral allograft leaflets. There were no significant differences between the two implantation methods. Annulus support of mitral allografts might be very useful in this type of operation and could prevent annular dilatation.

Hydrogen bonding effects on the (15)N and (1)H shielding tensors in nucleic acid base pairs.

The results of systematic ab initio calculations of (15)N and (1)H chemical shielding tensors in the GC base pair as a function of hydrogen bond length are presented for the first time. The hydrogen bond length characterized by the distance r(N...N) between purine N1 and pyrimidine N3 was varied between 2.57 and 3.50 A and the chemical shift tensors were calculated by the sum-over-states density functional perturbation theory. It is shown that the hydrogen bond length has a strong effect on the chemical shielding tensor of both imino proton and nitrogen, on their orientation, and, as a consequence, on the relaxation properties of both nuclei. For a nitrogen nucleus not involved in hydrogen bonding, the shielding tensor is nearly axially symmetric and almost collinear with the bond vector. As the length of the hydrogen bond decreases, the least shielding component sigma(11) deflects from the N-H vector and the shielding tensor becomes increasingly asymmetric. The significance of the presented results for the analysis of relaxation data and the efficiency of TROSY effects together with a summary of the relevant shielding parameters are presented and discussed.

Incorporation of carbon-14 in the biosynthesis of the macrolide antibiotic, LL-F28249-alpha.

A total of 76 mCi of 14C LL-F28249-alpha, nemadectin (1), having a specific activity of 35.2 microCi/mg was isolated from a fermentation using a mixture of approximately 600 mCi of 14C carboxyl labeled acetate, propionate and isobutyrate. Nemadectin was used to synthesize carbon-14 labeled moxidectin which is being developed as a highly efficient ectoparasitic anthelminth. The labeled positions were determined by 13C NMR analysis of 13C nemadectin which was obtained by similar incorporation of 13C carboxyl labeled acetate, propionate and isobutyrate.

Biosynthesis of the spermidine and guanidino units in the glycocinnamoylspermidine antibiotic cinodine.

The biosynthesis of the spermidine and guanidino groups has been studied with carbon-14 and carbon-13 labeled intermediates. Arginine, citrulline and ornithine are incorporated in good efficiency. The guanidino group of arginine and the ureido group of citrulline both label the guanidino group on the hexose sugar. None of the ureido groups in the antibiotic was enriched. It is likely that citrulline is converted to arginine before use in the biosynthesis. Arginine, citrulline and ornithine are incorporated as the four carbon unit of spermidine. All of the labeled 5 carbon from ornithine or from citrulline appears adjacent to the secondary amine in the four carbon unit of spermidine. This would indicate that unbound putrescine is not an immediate precursor of spermidine.

Biosynthesis of the glycocinnamoylspermidine antibiotic, cinodine.

The biosynthesis of cinodine from a combination of 14C- and 13C-labeled precursors has been investigated. Tyrosine was shown to be incorporated efficiently into the cinnamoyl moiety and glucosamine was found to be the origin of the three carbohydrate moieties. The relationship between the substrate dose and the enrichment of the labeled antibiotic has been elucidated so that it is possible to predict both the specific activity and the yield of the antibiotic obtained from the labeled substrates.

Biosynthetic origin of the carbon skeleton and oxygen atoms of the LL-F28249 alpha, a potent antiparasitic macrolide.

The biosynthesis of LL-F28249 alpha in a culture of Streptomyces cyaneogriseus has been studied using 13C, 14C and 18O labeled precursors. A complete 13C NMR spectrum of F28249 alpha has been assigned. Incorporation studies using 13C labeled precursors indicate that the carbon skeleton of F28249 alpha is derived from seven acetate, six propionate and one 2-methylpropionate units. The origin of the oxygen atoms of F28249 alpha has been examined by feeding [1-13C,18O2]acetate, [1-13C,18O2]propionate, [2-13C]acetate/18O2 and 18O2 separately to the fermentation culture and analyzing the resulting labeled LL-F28249 alpha samples by 13C NMR, electron impact MS and chemical ionization MS. Out of a total of eight oxygen atoms in LL-F28249 alpha, four oxygen atoms are derived from acetate, three from propionate and one from molecular oxygen.

Biosynthesis of the antibiotic maduramicin. Origin of the carbon and oxygen atoms as well as the 13C NMR assignments.

The biosynthesis of maduramicin alpha and beta in a culture of Actinomadura yumaensis has been studied using 13C, 14C and 18O labeled precursors. The alpha component of this recently discovered polyether antibiotic, containing forty-seven carbon atoms in a seven-ring system, is derived from eight acetate, seven propionate and four methionine molecules. The beta component which is missing one methoxy group incorporates three methionine methyl groups. The carbohydrate moiety was enriched by methionine, but not significantly by acetate or propionate. Studies of the incorporation of 13C labeled precursors permit the 13C NMR assignment of maduramicin. The origin of oxygen atoms of maduramicin has been examined by feeding [1-13C, 18O2]acetate and [1-13C, 18O2]propionate separately in the fermentation culture and the resulting doubly labeled maduramicin samples were analyzed by the isotopic shifts in the 13C NMR spectra. These results are consistent with the initial formation of a triene, which is converted to maduramicin by cyclization of the triepoxide.

Functional fluo-3/AM assay on P-glycoprotein transport activity in L1210/VCR cells by confocal microscopy.

Multidrug resistance (MDR) phenotype of L1210/VCR cell line, acquired by selection for vincristine (VCR), is predominantly mediated by P-glycoprotein (Pgp). Calcein/AM (Cal) was recently described as a fluorescent substrate for Pgp and may be used for measuring of transport activity of Pgp. Expression of Pgp in the cells prevents them to be loaded with the fluorescent marker. To detect the activity of Pgp, verapamil (Ver) or cyclosporine A (CsA) has to be used as Pgp inhibitors. Multidrug resistance protein (MRP), another drug efflux pump, may be inhibited by probenecid (Pro), i.e, the inhibitor of a wide variety of anion transporters. Ver, but not Pro, is able to induce the loading of L1210/CR cells by Cal that is measurable by fluorescence-activated cell sorter (FACS). Another dye, fluo-3/AM (F-3), has a similar behaviour like Cal. Using confocal microscopy we have proved that L1210/VCR cells, in contrast to parental sensitive cells, are not loaded with F-3. Marking of cells with the dye can be achieved using inhibitors of Pgp like Ver or CsA but not by Pro. These results indicate that F-3 is usable for detection of Pgp function in various MDR tissue cells.

[Personal experience with treatment of posttraumatic urethral distraction defects]. / Nase zkusenosti s lécbou posttraumatických uretrálních distrakcních defektu.

PURPOSE OF THE STUDY: Authors present their experience in the treatment of posttraumatic distraction urethral defect resulting from traumatic rupture of posterior urethra. MATERIAL: The group comprised 19 patients with posttraumatic urethral distraction defect (average age 41 year, range 27-65 years). In 16 of them (84%) resection urehtroplasty was performed and in three (16%) endoscopic internal urethrotomy was applied. The patients were evaluated of 19 to 48 months after surgery. METHOD: Urethroplasty was performed at least three months after the trauma, always under general anesthaesia in lithotomic position, using perinal approach. Dissection of bulbar urethra was followed by dissection and resection of fibrous posttraumatic distraction defect (the original membranous urethra). Prostatic apex and proximal end of lumbar urethra were spatulated and bulboprostatic anastomosis was performed restoring urethral continuity. A catheter was left in urethra for three weeks. In 12 patients it was necessary to separe corpora cavernosa addition and 5 patients required a wedge resection of the lower arch of public bones to allow urethral bridge the defect. Endoscopic internal urehtrotomy was also performed minimally three months after trauma, always on position 12 of the clock face opposite to symphysis with a discision of the whole stenotic part. Subsequently, catheter was inserted in urethra and left in place for four days. RESULTS: Resection urethroplasty as primary surgery was successful in 15 (94%) patients and only 1 patients (6%) required another reconstruction surgery. Endoscopic management was not successful in any patients (100%). Two of them (66%) had to undergo repeatedly a reconstruction surgery, the third one (33%) is regularly dilated. All patients after urethroplasty are under regular circumstances continent, only in two of them (13%) there occurs of urine in case of an extreme increase of abdominal pressure. Erectile function already impaired by the trauma did not worsen by the surgery in 4 patients (25%), in 2 patients (13%) with preoperatively normal erections there developed erectile dysfunction after urethroplasty of which in 1 patient a permanent disorder. The quality of life was in general evaluated by patients as excellent. DISCUSSION: Epicystotomy is a simple procedure ensuring urinary diversion in patients with posterior urethral rupture. However, such management of urethral rupture almost always results in the development posttraumatic distraction defect. Incontinence occurs in our group only in 2 (12%) patients, mainly in non-standard situations (gym, urgency). Night incontinence does not occur in our patients at all. Continence is in our patients ensured by lissosfincter which is fully sufficient. Erectile dysfunction may result from a trauma or a treatment. In our group all patients have a preserved erection prior to trauma and trauma was evident cause of the loss of erection only in 2 (12%) patients who were primarily treated by epicystotomy. In another 2 patients (12%) who were primarily treated after trauma for coincidental urinary bladder rupture it is impossible to state what caused the erectile dysfunction whether a fracture or surgery. In the acute phase during the revision of the rupture of posterior urethra the peroperative risk of the impairment of neurovascular bundles responsible for erection is much higher than in planned surgery. Satisfaction of patients with the treatment is reflected in the evaluation of the postoperative results and the quality of life in general. None of our patients managed by delayed internal urethrotomy was cured. One is regularly dilated, another two underwent urethroplasty. CONCLUSION: The technique of resection of urethral distraction defects with bulboprostatic anastomosis is a suitable way of the treatment of the preceding rupture of posterior urethra without impairement of continence or erection. A prerequisite of good results is a simple urine diversion by epicystostomy during the primary management of the posterior urethral rupture. Delayed endoscopic therapy of the distraction defect will not probably cure the patients but will result in regular dilatations. It may be an alternative treatment in polymorbid or biologically older patients.

Complex therapy of advanced colorectal carcinoma.

The present paper reports on a complex therapy of 18 patients with primary unresectable advanced carcinoma of the rectum and rectosigmoid. The results of surgery following complete chemoradiotherapy are evaluated. Radical surgery was successful in 15/18 patients. The authors describe a high incidence of postoperative complications and point out a high erudition of an oncosurgeon necessary for such intervention as well as for the indication of a patient to this extensive operation. (Tab. 2, Ref. 18.)

Urban growth and the world polity in the nineteenth and twentieth centuries: a research agenda.

PIP: The impact of political factors on urbanization is considered. The scope of the study is worldwide. The authors outline how political power, particularly the rise of the nation-state, can influence the growth and characteristics of urban systems. Comparisons are made between the nineteenth and twentieth centuries.^ieng